Use of a combination of myxoma virus and rapamycin for therapeutic treatment

ABSTRACT

The present invention relates to therapeutic use of a combination of Myxoma virus, including in combination with rapamycin. Treatment with rapamycin enhances the ability of Myxoma virus to selectively infect cells that have a deficient innate anti-viral response, including cells that are not responsive to interferon. The combination of rapamycin and Myxoma virus can be used to treat diseases characterized by the presence of such cells, including cancer. The invention also relates to therapeutic use of Myxoma virus that does not express functional M135R.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit and priority from U.S. provisionalpatent application No. 60/658,816, filed on Mar. 7, 2005, the contentsof which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to therapeutic use of Myxomavirus and rapamycin.

BACKGROUND OF THE INVENTION

Current treatments used to treat various types of cancer tend to work bypoisoning or killing the cancerous cell. Unfortunately, treatments thatare toxic to cancer cells typically tend to be toxic to healthy cells aswell. Moreover, the heterogenous nature of tumours is one of the primaryreasons that effective treatments for cancer remain elusive. Currentmainstream therapies such as chemotherapy and radiotherapy tend to beused within a narrow therapeutic window of toxicity. These types oftherapies are considered blunt tools that have limited applicability dueto the varying types of tumour cells and the limited window in whichthese treatments can be administered.

Modern anticancer therapies currently being developed attempt toselectively target tumour cells while being less toxic to healthy cells,thereby being more likely to leave healthy cells unaffected.

Oncolytic viral therapy is one approach that aims to exploit cellulardifferences between tumour cells and normal cells. This therapy usesreplication-competent, tumour-selective viral vectors as anti-canceragents. The oncolytic virus either specifically targets cancer cells forinfection, or is more suited for efficient replication in cancer cellsversus healthy cells. These replication-competent, oncolytic viruses areeither naturally occurring or genetically engineered to be a highlyselective and highly potent means of targeting the heterogeneous tumourpopulation. Since the replication selective oncolytic virus does notreplicate efficiently in normal cells, toxicity to the patient should below, particularly in comparison to traditional therapies such asradiation or chemotherapy.

Numerous studies have reported oncolytic activity for various virusstrains, with the most promising oncolytic viruses being a naturallyoccurring or genetically modified version of adenovirus, herpes simplexvirus 1 (“HSV1”), Reovirus, Vaccinia Virus, Vesicular Stomatitis Virus(“VSV”) or Poliovirus. Modified oncolytic viruses currently underinvestigation as anticancer agents include HSV, adenovirus, Newcastledisease virus (“NDV”), Reovirus and Vaccinia virus, measles, VSV andpoliovirus. Various oncolytic viruses are in Phase I and Phase IIclinical trials with some showing sustained efficacy. However, it isunknown which viruses will best fulfill the oncolytic goals of sustainedreplication, specificity and potent lytic activity. A completelyefficient candidate for an oncolytic viral vector would be one that hasa short lifecycle, forms mature virions quickly, spreads efficientlyfrom cell to cell and has a large genome ready for insertions. As well,evidence suggests that inhibiting the early innate immune response andslowing the development of Th1 responses are important for the efficacyof oncolytic therapy. It is clear that human viruses are highlyimmunogenic, as measured by the high level of antibody and T cellresponses that are observed in the normal population for many of theviruses being considered for the development of oncolytic viruses.

Clinical work has shown that current oncolytic viruses are indeed safe,but are not potent enough as monotherapies to be completely clinicallyeffective. As insufficient or inefficient infection of tumour cells isusually observed, the current movement is to arm candidate viruses bygenetically engineering them to express therapeutic transgenes toincrease their efficiency. Most of the above-mentioned oncolytic virusesare also being tested in combination with other common oncolytictherapies.

Adenovirus can be easily genetically manipulated and has well-knownassociated viral protein function. In addition, it is associated with afairly mild disease. The ONYX-015 human adenovirus (Onyx PharmaceuticalsInc.) is one of the most extensively tested oncolytic viruses that hasbeen optimized for clinical use. It is believed to replicatepreferentially in p53-negative tumours and shows potential in clinicaltrials with head and neck cancer patients. However, reports show thatONYX-015 has only produced an objective clinical response in 14% oftreated patients (Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M,Vokes E, Kuhn J, McCarty T, Landers S, Blackbum A, Romel L, Randlev B,Kaye S, Kim D. J. Clin. Oncol. 2001 Jan. 15; 19(2):289-98).

WO96/03997 and WO97/26904 describe a mutant oncolytic HSV that inhibitstumour cell growth and is specific to neuronal cells. Further advantagesare that the HSV can be genetically modified with ease, and drugs existto shut off any unwanted viral replication. However, the application ofsuch a common human pathogen is limited, as it is likely that thegeneral population has been exposed and acquired an immune response tothis virus, which would attenuate the lytic effect of the virus. HSV canalso cause serious side effects or a potentially fatal disease.

Reovirus type III is associated with relatively mild diseases and itsviral gene function is fairly well understood. Reovirus type III iscurrently being developed by Oncolytic Biotech as a cancer therapeuticwhich exhibits enhanced replication properties in cells expressingmutant ras oncogen and preferentially grows in PKR −/− cells (Strong J.E. and P. W. Lee, J. Virology, 1996. 70:612-616). However, Reovirus isdifficult to genetically manipulate and its viral replication cannot beeasily shut off.

VSV is associated with relatively mild diseases and also has well-knownviral gene function. WO99/04026 discloses the use of VSV as a vector ingene therapy for the expression of wide treatment of a variety ofdisorders. However, VSV suffers from the same problems as the Reovirusin that it is difficult to genetically manipulate and its viralreplication cannot be easily shut off.

Vaccina virus and Poliovirus are other candidate oncolytic virusesdescribed in the art but have been associated with a serious orpotentially fatal disease.

U.S. Pat. No. 4,806,347 discloses the use of gamma interferon and afragment of IFNγ against human tumour cells. WO99/18799 discloses amethod of treating disease in a mammal in which the diseased cells havedefects in an interferon-mediated antiviral response, comprisingadministering to the mammal a therapeutically effective amount of aninterferon-sensitive, replication competent clonal virus. Itspecifically discloses that VSV particles have toxic activity againsttumour cells but that alleviation of cytotoxicity in normal cells by VSVoccurs in the presence of interferon. WO99/18799 also discloses thatNDV-induced sensitivity was observed with the interferon-treated tumourcells but that adding interferon to normal cells makes these cellsresistant to NDV. This method aims to make cells sensitive to interferonby infecting them with interferon sensitive viruses.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected discovery that rabbitMyxoma virus, including a novel Myxoma virus that does not expressfunctional M135R protein, can selectively infect cells, including humantumour cells, that have a deficient innate anti-viral response,including those that are non-responsive to interferon, and that suchinfection is enhanced by treating such cells with the drug rapamycin.The term “innate” as used in this context describes non-antigen specificimmune response. Since Myxoma virus does not replicate efficiently innormal human cells, the virus can therefore be used as a treatment forvarious disorders and conditions characterized by cells that have adeficient innate anti-viral response, including cells that arenon-responsive to interferon, for example, as an oncolytic treatment forcancer. The virus can also be used to identify cells that have adeficient innate anti-viral response and to image these cells in vivo.

In one aspect, the present invention provides a method for inhibiting acell that has a deficient innate anti-viral response comprisingadministering to the cell an effective amount of a combination of Myxomavirus and rapamycin.

In one aspect, the invention provides a method for treating a diseasestate characterized by the presence of cells that have a deficientinnate anti-viral response, comprising administering to a patient inneed thereof an effective amount of a combination of Myxoma virus andrapamycin.

The present invention further provides use of an effective amount of acombination of Myxoma virus and rapamycin for inhibiting a cell that hasa deficient innate anti-viral response and for the manufacture of amedicament for inhibiting a cell that has a deficient innate anti-viralresponse.

The present invention further provides use of an effective amount of acombination of Myxoma virus and rapamycin for treating a disease statein a patient, wherein the disease state is characterized by the presenceof cells that have a deficient innate anti-viral response and for themanufacture of a medicament for treating such a disease state in apatient.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising Myxoma virus and rapamycin. The pharmaceuticalcomposition may be useful for inhibiting a cell that has a deficientinnate anti-viral response or for treating a disease state characterizedby the presence of cells that have a deficient innate anti-viralresponse.

In another aspect, the present invention provides a kit comprisingMyxoma virus, rapamycin and instructions for inhibiting a cell that hasa deficient innate anti-viral response or for treating a disease statecharacterized by the presence of cells that have a deficient innateanti-viral response. The disease states include cancer and a chronicviral infection.

The present invention further provides a method of detection a cell thathas a deficient innate anti-viral response, comprising exposing apopulation of cells to a combination of Myxoma virus and rapamycin;allowing the virus to infect a cell that has a deficient innateanti-viral response; and determining the infection of any cells of thepopulation of cells by the Myxoma virus.

The present invention is further based on the unexpected discovery thatrabbit Myxoma virus protein M135R is involved in eliciting an immuneresponse in rabbits and that a Myxoma virus strain that does not expressfunctional M135R can kill cells in vitro, but does not cause myxomatosisdisease in animals. Such a viral strain can be used to treat cellshaving a deficient innate anti-viral response, including those that arenon-responsive to interferon, and including treatments given incombination with the drug rapamycin, without the need for increasedcontainment of the virus, leading to improved safety.

In one aspect, the present invention provides a method for inhibiting acell that has a deficient innate anti-viral response comprisingadministering to the cell an effective amount of Myxoma virus that doesnot express functional M135R, optionally in combination with aneffective amount of rapamycin.

In one aspect, the invention provides a method for treating a diseasestate characterized by the presence of cells that have a deficientinnate anti-viral response, comprising administering to a patient inneed thereof an effective amount of Myxoma virus that does not expressfunctional M135R, optionally in combination with an effective amount ofrapamycin.

The present invention further provides use of an effective amount ofMyxoma virus that does not express functional M135R, optionally incombination with an effective amount of rapamycin, for inhibiting a cellthat has a deficient innate anti-viral response and in the manufactureof a medicament for inhibiting a cell that has a deficient innateanti-viral response.

The present invention further provides use of an effective amount ofMyxoma virus that does not express functional M135R, optionally incombination with an effective amount of rapamycin, for treating adisease state in a patient, wherein the disease state is characterizedby the presence of cells that have a deficient innate anti-viralresponse and in the manufacture of a medicament for treating such adisease state in a patient.

In a further aspect, the present invention provides a Myxoma virus thatdoes not express functional M135R.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising Myxoma virus that does not express functionalM135R. The pharmaceutical composition may be useful for inhibiting acell that has a deficient innate anti-viral response or for treating adisease state characterized by the presence of cells that have adeficient innate anti-viral response. The pharmaceutical composition mayfurther comprise rapamycin.

In another aspect, the present invention provides a kit comprisingMyxoma virus that does not express functional M135R and instructions forinhibiting a cell that has a deficient innate anti-viral response or fortreating a disease state characterized by the presence of cells thathave a deficient innate anti-viral response. The kit may furthercomprise rapamycin. The disease state includes cancer and a chronicviral infection.

The present invention further provides a method for detecting a cellthat has a deficient innate anti-viral response, comprising exposing apopulation of cells to a Myxoma virus that does not express functionalM135R, optionally in combination with rapamycin; allowing the virus toinfect a cell that has a deficient innate anti-viral response; anddetermining the infection of any cells of the population of cells by theMyxoma virus.

Other aspects and features of the present invention will become apparentto those of ordinary skill in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures. It should be understood, however, that thedetailed description and the specific examples while indicatingpreferred embodiments of the invention are given by way of illustrationonly, since various changes and modifications within the spirit andscope of the invention will become apparent to those skilled in the artfrom this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, which illustrate embodiments of the present invention,by way of example only,

FIG. 1 is a schematic diagram of an interferon mediated anti-viralsignalling scheme induced upon viral infection of a cell;

FIG. 2 is a phase contrast micrograph of nonpermissive WT murineembryonic fibroblasts (“MEFs”) after exposure to Myxoma virus,demonstrating that the MEFs become permissive after inhibition ofinterferon alp with neutralizing antibody;

FIG. 3 is a Western blot showing phosphorylation states (activation) ofSTAT1 and STAT2 after Myxoma virus infection, demonstrating thatnonpermissive infections of MEF cells is associated with activation ofSTAT 1 and STAT 2;

FIG. 4 is a Western blot showing phosphorylation states (inactivation)of STAT3, STAT4, STAT5 and STAT6 after Myxoma virus infection,demonstrating that nonpermissive infections of MEF cells does notactivate any of these species;

FIG. 5 is a phase contrast micrograph of IFNα/β R−/− MEFs and STAT1 −/−MEFs, IFNα/β R−/− MEFs and STAT1 −/− MEFs after infection with Myxomavirus, showing that inactivation of IFN/STAT/JAK signalling renderscells permissive for Myxoma infection;

FIG. 6 is a Western blot showing phosphorylation states of PKR innonpermissive wildtype MEFs after Myxoma virus infection, demonstratingthat PKR is not activated by Myxoma virus infection;

FIG. 7 is a Western blot showing phosphorylation states of PKR inwildtype MEFs either mock infected or pre-infected with Myxoma virus,showing that Myxoma virus blocks PKR activation in MEF cells;

FIG. 8 is a Western blot showing phosphorylation states of PERK inwildtype MEFs after Myxoma virus infection, demonstrating that Myxomavirus blocks PERK activation in MEF cells;

FIG. 9 is a phase contrast micrograph of PKR−/−, RNase L−/− and Mx1−/−triple knockout after exposure to Myxoma virus, showing that theantiviral state in MEF cells is mediated by a distinct pathway;

FIG. 10 is a phase contrast micrograph of PKR−/−, RNase L−/− and Mx1−/−triple knockout after exposure to Myxoma virus;

FIG. 11 is a phase contrast micrograph of PKR−/−, RNase L−/− and Mx1−/−triple knockout after treatment with neutralizing antibody to IFNα/β andafter exposure to Myxoma virus;

FIG. 12 is a Western blot showing phosphorylation levels of eIF2α andPKR in nonpermissive MEFs after treatment with neutralizing antibody toIFNα/β and after exposure to Myxoma virus, showing that eIF2αphosphorylation in nonresponsive cells is catalysed by a PKR-independentpathway;

FIG. 13 is a Western blot showing STAT1 phosphorylation states inPKR−/−, RNase L−/− and Mx1−/− triple knockout after Myxoma virusinfection, indicating normal IFN-induced signalling responses

FIG. 14 is a phase contrast micrograph illustrating subcellularlocalization of tyrosine-phosphorylated STAT1 in nonpermissivePKR−/−+RNaseL−/−+Mx1−/− cells at 12 hours post-infection, indicatingthat the activated STAT localizes to the nucleus, as predicted fornormal IFN/STAT signalling responses;

FIG. 15 is a fluorescent image of brains from nude mice havingintracranial gliomas mock-infected or infected with dead or live Myxomavirus expressing GFP, showing targeting of Myxoma to the glioma cells;

FIG. 16 is a fluorescent image and a photograph of a thin-sectionedmouse glioma infected with Myxoma virus expressing GFP showing that theMyxoma virus replicated only in tumour cells;

FIG. 17 is a phase contrast micrograph of HT29 human tumour cells,stained with either X-Gal or Crystal violet after infection with Myxomavirus, showing an example of a non-permissive infection in human cells;

FIG. 18 is a phase contrast micrograph of HOP92 human tumour cells,stained with X-Gal or Crystal Violet after infection with Myxoma virus,showing an example of a permissive infection of human cells;

FIG. 19 is phase contrast micrograph of OVCAR4 human tumour cells,stained with either X-Gal or Crystal Violet after infection with Myxomavirus, showing an example of a permissive infection of human cells;

FIG. 20 is a phase contrast micrograph of SK-MEL3 human tumour cells,stained with either X-Gal or Crystal Violet after infection with Myxomavirus, showing an example of a permissive infection of human cells;

FIG. 21 is a phase contrast micrograph of SK-MEL28 human tumour cells,stained with either X-Gal or Crystal Violet after infection with Myxomavirus, showing an example of a semi-permissive infection of human tumourcells;

FIG. 22 is a phase contrast micrograph of BGMK cells, stained witheither X-Gal or Crystal Violet after infection with Myxoma virus,showing a typical permissive control infection;

FIG. 23 is a phase contrast micrograph of positive control BGMK cellsand human tumour lines U87, A172 and U373 infected with increasingconcentrations of Myxoma virus expressing the LacZ protein, stained withX-Gal, showing that these human glioma cells were all permissive forMyxoma virus replication;

FIG. 24 is a graph depicting survival rate of BGMK, U87, A172 and U373cells infected with Myxoma virus, 72 hours post-infection, at increasingconcentrations of the virus, demonstrating the ability of Myxoma to killall of these cells;

FIG. 25 is a phase contrast micrograph and fluorescence micrograph ofSF04 1585 astrocytoma cells infected with MV GFP, showing the infectionin primary human glioma cells;

FIG. 26 is a phase contrast micrograph of U373 glioma cells infectedwith Myxoma virus expressing the LacZ protein and stained with X-Gal,showing infection of these human tumour cells;

FIG. 27 is a graph depicting the survival rate of SF04 1585 cellsinfected with MV GFP 48 hours post-infection, showing killing of theseinfected human tumour cells;

FIG. 28 is a fluorescence micrograph of Daoy and D384 medulloblastomalines infected with Myxoma virus expressing GFP, showing infection ofthese human tumour cells.

FIG. 29 is graphical representations of the rate of virus production invarious cell lines with or without pre-treatment with rapamycin: BGMK(primate control cell line); RK-13 and RL5 (rabbit control cell lines);4T1 and B16F10 (mouse cancer cell lines); HOS, PC3, 786-0, HCT116, ACHN,MCF-7, M14 and COLO205 (human cancer cell lines); using wildtype virusvMyxLac and the M-T5 knock out virus vMyxT5KO as indicated;

FIG. 30 is photographs of virally infected cell lines, infected witheither vMyxLac or vMyxLacT5-;

FIG. 31 is graphical representations of the rate of virus production invarious cell lines (BGMK; A9; MCF-7; MDA-MB-435; M14; and COLO205) withor without pre-treatment with rapamycin;

FIG. 32 is (A) a schematic alignment of Myxoma virus protein M135R andVaccinia virus protein B18R and (B) an amino acid sequence alignmentbetween M135R and the first 179 amino acids of B 18R;

FIG. 33 is (A) a Western blot of M135R expressed in BGMK cells infectedwith Myxoma virus Lausanne (vMyxLau) and (B) a Western blot of M135Rexpressed in BGMK cells infected with vMyxLau and treated with araC,tunicamycin or monensin;

FIG. 34 is (A) a fluorescence micrograph of BGMK cells mock infected orinfected with Myxoma virus and stained for M135R and (B) a Western blotagainst immunoprecipitations or cell lysates of cells infected withwildtype Myxoma virus (vMyxgfp) or an M135R knockout strain (vMyx135KO)using anti-M135R antibody;

FIG. 35 is (A) is a schematic diagram of the cloning strategy to producevMyx135KO, (1) an agarose gel of the PCR insert product and (C) aWestern blot of cells infected with wildtype and M135R knockout Myxomavirus;

FIG. 36 is a growth curve of viral foci in BGMK cells infected withvMyxgfp or vMyx135KO;

FIG. 37 is light and fluorescent micrographs of rabbit embryofibroblasts infected with vMyxgfp or vMyx135KO;

FIG. 38 is light and fluorescent micrographs of rabbit HIG82 fibroblastsinfected with vMyxgfp or vMyx135KO;

FIG. 39 is light and fluorescent micrographs of human primaryfibroblasts infected with vMyxgfp or vMyx135KO;

FIG. 40 is a graph of body temperature in rabbits infected with vMyxLauor vMyx135KO ;

FIG. 41 is a graph of ¹²⁵I emissions of cells mock infected or infectedwith vMyxgfp or vMyx135KO and treated with ¹²⁵I-labelled rabbitinterferon α/β;

FIG. 42 is a graph of foci formed by infecting RK13 or BGMK cells withvMyxgfp or vMyx135KO, in which cells were untreated or treated withrabbit interferon α/β 24 hours prior to infection; and

FIG. 43 is photographs of Western blots using cell lysates from 786-0human cancer cells that were pre-treated with either 20 nM rapamycin (R)or with the vehicle control (D), probed using antibodies directedagainst the indicated proteins.

DETAILED DESCRIPTION

Previously, the inventors have discovered that wildtype Myxoma virus, avirus that normally infects rabbits, can selectively infect and killcells, including human cells, that have a deficient innate anti-viralresponse, for example, cells that are non-responsive to interferon, asdescribed in the application PCT/CA2004/000341, which is herein fullyincorporated by reference. Myxoma virus does not replicate efficientlyin normal human cells. Since many diseases or conditions arecharacterized by the presence of cells that have a deficient innateanti-viral response, including cells that are not responsive tointerferon, for example, cancer, Myxoma virus can be used to treat suchdiseases and conditions, including cancer, with low toxicity for normalhealthy cells. Myxoma virus can also be used to treat chronicallyinfected cells as such cells have a deficient innate anti-viralresponse. For example, many viruses encode gene products that functionto inhibit the antiviral, interferon response of cells; Myxoma virus canselectively infect such cells.

Myxoma virus (“MV”) is the causative agent of myxomatosis in rabbits. MVbelongs to the Leporipoxvirus genus of the Poxyiridae family, thelargest of the DNA viruses. MV induces a benign disease in its naturalhost, the Sylvilagus rabbit in the Americas. However, it is a virulentand host-specific poxvirus that causes a fatal disease in Europeanrabbits, characterized by lesions found systemically and especiallyaround the mucosal areas. (Cameron C, Hota-Mitchell S, Chen L, BarrettJ, Cao J X, Macaulay C, Willer D, Evans D, McFadden G. Virology 1999,264(2): 298-318; Kerr P & McFadden G. Viral Immunology 2002, 15(2):229-246).

MV is a large virus with a double-stranded DNA genome of 163 kb whichreplicates in the cytoplasm of infected cells (B. N. Fields, D. M.Knipe, P. M. Howley, Eds., Virology Lippincott Raven Press, New York,2nd ed., 1996). MV is known to encode a variety of cell-associated andsecreted proteins that have been implicated in down-regulation of thehost's immune and inflammatory responses and inhibition of apoptosis ofvirus-infected cells. MV can be taken up by all human somatic cells.However, other than in normal somatic rabbit cells, if the cells have anormal innate anti-viral response, the virus will not be able toproductively infect the cell, meaning the virus will not be able toreplicate and cause cell death.

Interferons (“IFNs”) are a family of cytokines that are secreted inresponse to a variety of stimuli. Interferons bind to cell surfacereceptors, activating a signaling cascade that leads to numerouscellular responses, including an anti-viral response and induction ofgrowth inhibition and/or apoptotic signals. Interferons are classifiedas either type I or type II. Type I IFNs include IFN-α,-β,-τ, and -ω),which are all monomeric; the only type II IFN is IFN-γ, a dimer. Twelvedifferent subtypes of IFN-α are produced by 14 genes, but all other IFNsare monogenic (Arduini et al., 1999). IFNs exert direct anti-tumouractivity via the modulation of oncogene expression. Overexpression ofgrowth-stimulating oncogenes or loss of tumour suppressor oncogenes canlead to malignant transformation. Some oncogenes implicated in thegenesis of cancer are p53, Rb, PC, NF1, WT1, DCC.

Myxoma virus, as well as other oncolytic viruses such as Reovirus andVSV, needs to bypass the anti-viral defenses that exist in normalhealthy cells in order to be able to replicate within cells. MV andother oncolytic viruses induce interferon production, and are generallysensitive to the anti-viral effect of the IFN pathway. Relevant proteinsinduced by the IFN anti-viral response, and which principally affectvirus multiplication include PKR, OAS synthetase and Rnase L nuclease.PKR activates eIF2α, leading to inhibition of translation and inductionof apoptosis. A schematic representation of the IFN response pathway isdepicted in FIG. 1. In normal cells, MV is directly affected by PKR andeIF2α.

Anti-viral response pathways are often disrupted in cancerous cells. Forexample, reduced or defective response to IFN is a genetic defect thatoften arises during the process of transformation and tumour evolution.Over 80% of tumour cell lines do not respond to, or exhibit impairedresponses to, interferon. (Stojdl et al., Cancer Cell (2003) 4: 263-275and references cited therein; Wong et al. J Biol. Chem. (1997)272(45):28779-85; Sun et al. Blood. (1998) 91(2):570-6; Matin et al.Cancer Res. (2001) 61(5):2261-6; Balachandran et al Cancer Cell (2004)5(1):51-65). As previously disclosed in PCT/CA2004/000341, MV can infectand kill cancer cells, including human tumour cells, and without beinglimited by any particular theory, it is believed that MV can infectthese cells because they have a deficient innate anti-viral response.

Evidence suggests that inhibiting the early innate immune response andslowing the development of Th1 responses are important for the efficacyof oncolytic therapy. Although Myxoma virus is a virulent virus, it ishost-specific and has a very narrow host range; it does not infecthumans or mice. Without being limited by any specific theory, it isbelieved that since Myxoma virus is a non-human virus, it shouldencounter no pre-existing immune recognition in humans. Therefore, itspotential as an oncolytic virus will be less compromised and Myxomavirus should provide more potent infection of permissive tumour cellsthan native human viruses, and thereby can provide an effectiveoncolytic treatment for cancer.

The Myxoma virus host range gene M-T5 appears to play a critical roleduring Myxoma virus infection of many human tumour cell lines (Sypula etal, (2004) Gene Ther. Mol. Biol. 8:103). The MT-5 gene encodes anankyrin repeat protein that is required for Myxoma replication in rabbitlymphocytes, and Myxoma virus with the MT-5 gene deleted cannot causemyxomatosis in susceptible rabbits (Mossman et al, (1996) J. Virol. 70:4394). Available evidence suggests that differences in the intracellularsignalling within an infected human tumour cell are critical fordistinguishing human tumour cells that are permissive to Myxoma virusinfection and productive replication (Johnston et al, (2003) J. Virol.77: 5877).

Furthermore, Myxoma virus possesses a protein, M135R, which displayshomology to the amino terminus portion of interferon α/β receptor(“IFNα/β-R”). It has been suggested that M135R mimics the host IFNα/β-Rin order to prevent IFNα/β from triggering a host anti-viral response(Barrett et al., Seminars in Immunology (2001)13:73-84). The predictionis based on sequence homology to the viral IFNα/β-R from vaccinia virus,B18R, and it has been demonstrated that Vaccinia virus (“VV”) employssuch an immune evasion strategy. However, M135R is only half the size ofVV B18R and all other IFNα/β-R homologs from sequenced poxviruses, andin all cases aligns only to the amino terminus half of the homolog.

The inventors have discovered that even though immunofluorescenceresults suggest that M135R localizes to the cell surface, attempts todemonstrate the ability of M135R to interact with IFNα/β have beennegative. Despite these results, the inventors have discovered thatdeletion of M135R severely attenuates the ability of Myxoma virus tocause disease in host animals although Myxoma virus having such adeletion is equally effective at infecting and killing cells in vitrocompared to wildtype MV. Thus, in one aspect, the present inventionrelates to the discovery that Myxoma virus that does not expressfunctional M135R is useful for treatment of cells having a deficientinnate anti-viral response, including for oncolytic studies, since thisvirus provides a safer alternative for oncolytic viral therapy as nounusual containment strategies should be needed for patients undergoingtreatment.

In another aspect, the present invention relates to the discovery thatthe anti-cancer agent rapamycin acts to enhance the levels ofinfectivity of Myxoma virus in human tumour cells which are permissivefor Myxoma virus infection, and that rapamycin allows replication ofcertain strains of Myxoma virus in human tumour cells which, withoutrapamycin, are restrictive for the replication of those strains ofMyxoma virus. A cell that is permissive for Myxoma virus infection is acell that the virus can enter and in which the virus can productivelyreproduce. Permissive cells may have defects or mutations in one or moreof the pathways that involve the proteins PTEN, PDK, AKT, GSK, Raf, mTORor P70S6K. A restrictive cell is a cell which is permissive to Myxomavirus only under certain conditions, but does not allow productiveinfection under other conditions. For example, a restrictive cell may bepermissive to wildtype strains of the virus, but does not allow certainmutant Myxoma strains, for example a strain having the MT-5 gene knockedout, to productively reproduce. In another example, a cell restrictivefor Myxoma virus may not permit productive infection of Myxoma virusalone, but when treated with rapamycin, the same Myxoma virus is able toproductively infect the cell. Abortive cell lines are non-permissive forMyxoma virus infection, meaning that the virus may be able to enter thecell, but does not productively infect the cell.

Thus, rapamycin, when used in combination with Myxoma virus, enhancesthe infectivity of Myxoma virus for cells having a deficient innateanti-viral response. The present invention relates to the use ofrapamycin in combination with Myxoma virus to treat cells having adeficient innate anti-viral response.

Rapamycin is a macrocyclic lactone that has been shown to be the activeantifungal compound purified from the soil bacterium Streptonzyceshygroscopicus. Rapamycin as used herein refers to rapamycin (alsoreferred to as sirolimus) and analogs or derivatives thereof capable ofcomplexing with FKBP12 and inhibiting mTOR, including the analogsCCI-779 (also referred to as cell cycle inhibitor-779 orrapamycin-42,2,2-bis(hydroxymethyl)-propionic acid) and RAD001 (alsoreferred to as everolimus or 40-O-(2-hydroxyethyl)-rapamycin).Rapamycin, CCI-779 and RAD001 are commercially available, and rapamycinis available under the name Rapamune™, from Wyeth-Ayerst. The termrapamycin further includes pharmaceutically acceptable salts and estersof rapamycin, its hydrates, solvates, polymorphs, analogs orderivatives, as well as pro-drugs or precursors which are metabolized orconverted to rapamycin or its analogs or derivatives during use, forexample when administered to a patient.

Rapamycin as an inhibitor of cellular signaling is highly specific: itenters the cell and binds to a cellular protein known as FKBP12. Therapamycin/FKBP12 complex then binds to the specific cellular target mTOR(mammalian Target of Rapamycin). Many cancers have been shown to developfrom an over activity of signaling molecules such as P13K, or a loss ofthe tumor suppressor gene PTEN. Both of these molecules lie upstream ofmTOR. mTOR has been shown to be a central regulator of cellproliferation, growth, differentiation, migration and survival, and istherefore an ideal target in stemming the uncontrolled growth of cancercells. Cancer cell lines that are sensitive to rapamycin are generallythose that have resulted from an activation of the pathway through mTOR.

Rapamycins are used primarily in transplant patients as an alternativeor complementary treatment to cyclosporine treatment. In transplantpatients, rapamycin treatment generally has fewer side effects thatcyclosporine A or FK506. In addition, retrospective studies haveindicated that patients on rapamycin treatment generally develop fewercancers and have a lower incidence of CMV (cytomegalovirus; a herpesvirus) infection. It is therefore surprising that rapamycin treatmentenhances Myxoma virus infection of cancer cells, particularly in lightof research postulating that CMV replication should be reduced byrapamycin (reviewed by Ponticelli: “The pleiotropic effects of mTORinhibitors” in J Nephrology 2004; 17: 762). Without being limited to aparticular theory, it is possible that Myxoma virus takes advantage ofaberrant signaling through the mTOR pathway that may be associated withthe neoplastic phenotype of these cells. Manipulation of this pathway bymTOR inhibitors could then be a selective advantage to the virus.

Thus, there is provided a method for inhibiting a cell that has adeficient innate anti-viral response comprising administering to thecell an effective amount of Myxoma virus. In a further embodiment, thevirus is administered in combination with an effective amount ofrapamycin.

The Myxoma virus may be any virus that belongs to the Leporipoxvirusspecies of pox viruses that is replication-competent. The Myxoma virusmay be a wild-type strain of Myxoma virus or it may be a geneticallymodified strain of Myxoma virus, including an MT-5 knockout strain ofMyxoma. The Myxoma virus may be a strain that has an attenuated affectin rabbits, thereby causing lower risk of disease, including a strainthat does not express functional M135 protein, as described below.

In a particular embodiment, the Myxoma virus is a Myxoma virus that doesnot express functional M135R.

A Myxoma virus that does not express functional M135R includes a Myxomavirus that has part, or all, of the open reading frame that encodesM135R deleted, replaced or interrupted such that no gene product, nostable gene product, or no functional gene product is expressed. Such avirus also includes a Myxoma virus that has part, or all, of the M135Rgene regulatory region deleted, replaced or interrupted such that noprotein can be expressed from the gene encoding M135R. Functional M135Rprotein is M135R that is transcribed, translated, folded,post-translationally modified and localized within the cell, and whichallows Myxoma virus to cause myxomatosis in an infected host. If theM135R protein is not, or not properly or not sufficiently, transcribed,translated, folded, post-translationally modified or localized withinthe cell such that an infected host does not develop myxomatosis, thenno functional M135R protein is expressed in the cell.

In a further embodiment, the cell is non-responsive to interferon.

In specific embodiments, the cell is a mammalian cancer cell. In oneembodiment the cell is a human cancer cell including a human solidtumour cell.

In another embodiment, the cell is chronically infected with a virus.

A “combination” of rapamycin and Myxoma virus for administration may beformulated together in the same dosage form or may be formulated inseparate dosage forms, and the separate dosage forms may be the sameform or different forms, for administration by the same mode or bydifferent modes of administration. Furthermore, administration of acombination of rapamycin and Myxoma virus, when not together in the samedosage form, means that the rapamycin and Myxoma virus are administeredconcurrently to the mammal being treated, and may be administered at thesame time or sequentially in any order or at different points in time.Thus, rapamycin and Myxoma virus may be administered separately butsufficiently closely in time so as to provide the desired therapeuticeffect.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desired result.

The term “a cell that has a deficient inmate anti-viral response” asused herein refers to a cell that, when exposed to a virus or wheninvaded by a virus, does not induce anti-viral defense mechanisms, whichinclude inhibition of viral replication, production of interferon,induction of the interferon response pathway, and apoptosis, which mayor may not be mediated by interferon, and is thereby infectable by MV,alone or in combination with rapamycin treatment. The term includes acell that has a reduced or defective innate anti-viral response uponexposure to or infection by a virus as compared to a normal cell, forexample, a non-infected, or non-cancer cell. This includes a cell thatis non-responsive to interferon and a cell that has a reduced ordefective apoptotic response or induction of the apoptotic pathway. Thedeficiency may be caused by various causes, including infection, geneticdefect, or environmental stress. It will however be understood that whenthe deficiency is caused by a pre-existing infection, superinfection byMV may be excluded and a skilled person can readily identify suchinstances. A skilled person can readily determine without undueexperimentation whether any given cell type has a deficient innateanti-viral response and therefore infectable by Myxoma virus, eitheralone or in combination with rapamycin treatment. For example, VSV iscommonly used to measure an anti-viral response of a cell.

To assess whether a given cell type, for example a given cancer celltype, has a deficient innate anti-viral response, a skilled person cantake an explant, grow some of the cells in vitro and determineinfectability by VSV or alternatively, by Myxoma virus, including Myxomavirus in combination with rapamycin.

The term “a cell that is non-responsive to interferon” as usedthroughout the specification means a cell that does not respond to theactivity of interferon, for example anti-viral or anti-tumour activityof interferon or that has an abnormal interferon response, for example,a reduced or ineffective response to interferon, or abnormal interferonsignalling as measured by, for example, phosphorylation or activation ofsignalling molecules such as transcription factors, for example STAT1.For example, without limitation, the cell may not undergo inhibition ofproliferation or it may not be killed when exposed to interferon levelssufficient to induce such a response in a cell that is responsive tointerferon. The cell that is non-responsive to interferon may have adefect in the intracellular signalling pathway or pathways that arenormally activated in the responsive cells. Typically, susceptibility toinfection by VSV is indicative of non-responsiveness to interferon, anda skilled person can readily determine whether a particular cell isnon-responsive to interferon by its ability, or lack thereof, to inhibitVSV infection in the presence of interferon or using other markers ofinterferon activity known in the art, for example, the level ofexpression of IFN stimulated genes such as PKR, STAT, OAS, MX.

The term “replication-competent” as used throughout the specificationrefers to a virus that is capable of infecting and replicating within aparticular host cell. This includes a virus which alone is restrictedfor replication in a particular host cell, but when the host cell istreated with rapamycin, the virus can then productively infect thatcell.

The term “a cell” as used herein includes a single cell as well as aplurality or population of cells. Administering an agent to a cellincludes both in vitro and in vivo administrations.

The term “animal” as used herein includes all members of the animalkingdom, including particularly mammals, especially humans.

The term “inhibiting” a cell that has a deficient innate anti-viralresponse includes cell death by lysis or apoptosis or other mechanismsof cell death, in addition to rendering the cell incapable of growing ordividing or reducing or retarding cell growth or division.

The Myxoma virus genome may be readily modified to express one or moretherapeutic transgenes using standard molecular biology techniques knownto a skilled person, and described for example in Sambrook et al.((2001) Molecular Cloning: a Laboratory Manual, 3^(rd) ed., Cold SpringHarbour Laboratory Press). A skilled person will be able to readilydetermine which portions of the Myxoma viral genome can be deleted suchthat the virus is still capable of productive infection. For example,non-essential regions of the viral genome that can be deleted can bededuced from comparing the published viral genome sequence with thegenomes of other well-characterized viruses (see for example C. Cameron,S. Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay, D.Willer, D. Evans, and G. McFadden, Virology (1999) 264: 298-318)).

The term “therapeutic gene” or “therapeutic transgenes” as used hereinis intended to describe broadly any gene the expression of which effectsa desired result, for example, anti-cancer effect. For example, thevirus may be modified to carry a gene that will enhance the anti-cancereffect of the viral treatment. Such a gene may be a gene that isinvolved in triggering apoptosis, or is involved in targeting theinfected cell for immune destruction, such as a gene that repairs a lackof response to interferon, or which results in the expression of a cellsurface marker that stimulates an antibody response, such as a bacterialcell surface antigen. The virus may also be modified to express genesinvolved in shutting off the neoplastic or cancer cell's proliferationand growth, thereby preventing the cells from dividing. As well, thevirus may be modified to include therapeutic genes, such as genesinvolved in the synthesis of chemotherapeutic agents, or it may bemodified to have increased replication levels in cells of the particularspecies from which the cells to be inhibited or killed are derived, forexample, human cells. Specific examples of genes that may be insertedinto the Myxoma virus to increase its anti-cancer effect include thehuman gene for the TRAIL protein or the adenoviral gene that encodes theE4 or f4 polypeptide, both of which proteins are involved in killinghuman tumour cells.

It will be understood that therapeutic effect of the Myxoma virus,including when used in combination with rapamycin, may be achieved bycell lysis by the virus or by delivery of therapeutic products by thevirus. The inclusion of rapamycin in combination with the Myxoma virusshould allow for enhancement of the effect of Myxoma virus alone. Thatis, the Myxoma virus, when administered in combination with rapamycinshould be able to productively infect a greater number of target cellsthan Myxoma virus alone, or should be able to productively infect targetcells having a deficient innate anti-viral response which arerestrictive for productive infection by Myxoma virus in the absence ofrapamycin.

The virus may be prepared using standard techniques known in the art.For example, the virus may be prepared by infecting cultured rabbitcells with the Myxoma virus strain that is to be used, allowing theinfection to progress such that the virus replicates in the culturedcells and can be released by standard methods known in the art fordisrupting the cell surface and thereby releasing the virus particlesfor harvesting. Once harvested, the virus titre may be determined byinfecting a confluent lawn of rabbit cells and performing a plaque assay(see Mossman et al. (1996) Virology 215:17-30).

There is also provided a method for treating a disease statecharacterized by the presence of cells that have a deficient innateanti-viral response in a patient in need of such treatment comprisingadministering to the patient an effective amount of Myxoma virus,optionally in combination with rapamycin. The patient may be any animal,including a mammal, including a human.

“A disease state characterized by the presence of cells that have adeficient innate anti-viral response” as used herein refers to anydisease, disorder or condition which is associated with, related to, ora characteristic of which is, the presence of cells that have adeficient innate anti-viral response and which disease, disorder,condition or symptoms thereof may be treated by killing these cells. Forexample, the disease state may be cancer. The disease state may alsoinclude chronic infection with a virus.

“Treating” a disease state refers to an approach for obtainingbeneficial or desired results, including clinical results. Beneficial ordesired clinical results can include, but are not limited to,alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilization of the state ofdisease, prevention of development of disease, prevention of spread ofdisease, delay or slowing of disease progression, delay or slowing ofdisease onset, amelioration or palliation of the disease state, andremission (whether partial or total). “Treating” can also meanprolonging survival of a patient beyond that expected in the absence oftreatment. “Treating” can also mean inhibiting the progression ofdisease, slowing the progression of disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently.

In one embodiment, the disease state is cancer. The cancer may be anytype of cancer wherein at least some of the cells, although notnecessarily all of the cells have a deficient innate anti-viralresponse. In one embodiment, the cancer may be a cancer wherein at leastsome of the cells are non-responsive to interferon. As used herein, theterms “tumour”, “tumour cells”, “cancer” and “cancer cells”, (usedinterchangeably) refer to cells that exhibit abnormal growth,characterized by a significant loss of control of cell proliferation orcells that have been immortalized. The term “cancer” or “tumour”includes metastatic as well as non-metastatic cancer or tumours. As usedherein, “neoplastic” or “neoplasm” broadly refers to a cell or cellsthat proliferate without normal growth inhibition mechanisms, andtherefore includes benign tumours, in addition to cancer as well asdysplastic or hyperplastic cells.

A cancer may be diagnosed using criteria generally accepted in the art,including the presence of a malignant tumor.

Types of cancer that may be treated according to the present inventioninclude, but are not limited to, hematopoietic cell cancers includingleukemias and lymphomas, colon cancer, lung cancer, kidney cancer,pancreas cancer, endometrial cancer, thyroid cancer, oral cancer,ovarian cancer, laryngeal cancer, hepatocellular cancer, bile ductcancer, squamous cell carcinoma, prostate cancer, breast cancer,cervical cancer, colorectal cancer, melanomas and any other tumours.Solid tumours such as sarcomas and carcinomas include but are notlimited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing'stumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoidmalignancy, pancreatic cancer, breast cancer, lung cancers, ovariancancer, prostate cancer, hepatocellular carcinoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cellcarcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor,cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

In another embodiment, the disease state is a chronic viral infection.

The chronically infecting virus may be any virus that infects andreplicates in cells of an animal in a persistent manner over a prolongedperiod so as to cause a pathological condition. The chronicallyinfecting virus may be a virus that is associated or correlated with thedevelopment of cancer.

A chronic infection with a virus may be diagnosed using standard methodsknown in the art. For example, a chronic viral infection may be detectedby the presence of anti-viral antibodies in the patient or a positivetest for the presence of viral RNA or DNA in cells of the patient.

When administered to a patient, an effective amount of the Myxoma virus,and optionally the combination of Myxoma virus with rapamycin, is theamount required, at the dosages and for sufficient time period, for thevirus to alleviate, improve, mitigate, ameliorate, stabilize, preventthe spread of, slow or delay the progression of or cure the disease. Forexample, it may be an amount sufficient to achieve the effect ofreducing the number of or destroying cancerous cells or neoplasticcells, or reducing the number of or destroying cells chronicallyinfected with a virus, or inhibiting the growth and/or proliferation ofsuch cells.

The effective amount to be administered to a patient can vary dependingon many factors such as the pharmacodynamic properties of the Myxomavirus and the optionally rapamycin, the modes of administration, theage, health and weight of the patient, the nature and extent of thedisease state, the frequency of the treatment and the type of concurrenttreatment, if any, and the virulence and titre of the virus.

One of skill in the art can determine the appropriate amount of Myxomavirus for administration based on the above factors. The virus may beadministered initially in a suitable amount that may be adjusted asrequired, depending on the clinical response of the patient. Theeffective amount of virus can be determined empirically and depends onthe maximal amount of the virus that can be administered safely, and theminimal amount of the virus that produces the desired result.

Myxoma virus may be administered to the patient using standard methodsof administration. In one embodiment, the virus is administeredsystemically. In another embodiment, the virus is administered byinjection at the disease site. In a particular embodiment, the diseasestate is a solid tumour and the virus is administered by injection atthe tumour site. In various embodiments, the virus may be administeredorally or parenterally, or by any standard method known in the art.

To produce the same clinical effect when administering the virussystemically as that achieved through injection of the virus at thedisease site, administration of significantly higher amounts of virusmay be required. However, the appropriate dose level should be theminimum amount that would achieve the desired result.

The concentration of virus to be administered will vary depending on thevirulence of the particular strain of Myxoma that is to be administeredand on the nature of the cells that are being targeted. In oneembodiment, a dose of less than about 10⁹ plaque forming units (“pfu”)is administered to a human patient. In various embodiments, betweenabout 10² to about 10⁹ pfu, between about 10² to about 10⁷ pfu, betweenabout 10³ to about 10⁶ pfu, or between about 10⁴ to about 10⁵ pfu may beadministered in a single dose.

One of skill in the art can also determine, using the above factors, theappropriate amount of rapamycin to administer to a patient. Theeffective amount of rapamycin can be determined empirically and willdepend on the amount and strain of virus being administered, the maximumamount of rapamycin that can be safely administered and the minimalamount of rapamycin that can be administered in order to achieve anenhancement of the infectivity of Myxoma virus.

Rapamycin may be administered to the patient using standard methods ofadministration. In one embodiment, the rapamycin is administeredsystemically. In another embodiment, the rapamycin is administered byinjection at the disease site. In a particular embodiment, the diseasestate is a solid tumour and the rapamycin is administered by injectionat the tumour site. In various embodiments, the rapamycin may beadministered orally or parenterally, or by any standard method known inthe art.

The total amount of rapamycin may be administered in a single dose or inmultiple doses spread out over 1 day or several days. The frequency andduration of administration of doses can be readily determined. Theschedule of dosing will depend on the length of time that the Myxomavirus is to be administered. For example, rapamycin may be administeredonce to a patient, or may be administered 2 to 4 times per day.

In various embodiments, the dose of rapamycin may be from about 0.01 toabout 250 mg per kg of body weight per day, from about 0.01 to 50 mg perkg of body weight per day, from about 0.05 to 10 mg per kg of bodyweight per day, or from about 0.1 to 7.5 mg per kg of body weight perday.

Effective amounts of a combination of Myxoma virus and rapamycin can begiven repeatedly, depending upon the effect of the initial treatmentregimen. Administrations are typically given periodically, whilemonitoring any response. It will be recognized by a skilled person thatlower or higher dosages than those indicated above may be given,according to the administration schedules and routes selected.

The Myxoma virus, optionally in combination with rapamycin, may beadministered as a sole therapy or may be administered in combinationwith other therapies, including chemotherapy, radiation therapy or otheranti-viral therapies. For example, the Myxoma virus, optionally incombination with rapamycin, may be administered either prior to orfollowing surgical removal of a primary tumour or prior to, concurrentlywith or following treatment such as administration of radiotherapy orconventional chemotherapeutic drugs. In one embodiment, the Myxomavirus, optionally in combination with rapamycin can be administered incombination with, or in a sequential fashion with, other oncolyticviruses, which may demonstrate specificity for varying tumour celltypes.

To aid in administration, the Myxoma virus, optionally in combinationtogether with rapamycin, may be formulated as an ingredient in apharmaceutical composition. Therefore, in a further embodiment, there isprovided a pharmaceutical composition comprising Myxoma virus, andoptionally rapamycin, and a pharmaceutically acceptable diluent. Theinvention in one aspect therefore also includes such pharmaceuticalcompositions for use in inhibiting a cell that has a deficient innateanti-viral response or treating a disease state characterized by thepresence of cells that have a deficient innate anti-viral response. Thecompositions may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives and variouscompatible carriers. For all forms of delivery, the recombinant Myxomavirus may be formulated in a physiological salt solution.

The pharmaceutical compositions may additionally contain additionaltherapeutic agents, such as additional anti-cancer agents. In oneembodiment, the compositions include a chemotherapeutic agent. Thechemotherapeutic agent, for example, may be substantially any agentwhich exhibits an oncolytic effect against cancer cells or neoplasticcells of the patient and that does not inhibit or diminish the tumourkilling effect of the Myxoma virus. For example, the chemotherapeuticagent may be, without limitation, an anthracycline, an alkylating agent,an alkyl sulfonate, an aziridine, an ethylenimine, a methylmelamine, anitrogen mustard, a nitrosourea, an antibiotic, an antimetabolite, afolic acid analogue, a purine analogue, a pyrimidine analogue, anenzyme, a podophyllotoxin, a platinum-containing agent or a cytokine.Preferably, the chemotherapeutic agent is one that is known to beeffective against the particular cell type that is cancerous orneoplastic.

The proportion and identity of the pharmaceutically acceptable diluentis determined by chosen route of administration, compatibility with alive virus, and where applicable compatibility with the chemicalstability of rapamycin, and standard pharmaceutical practice. Generally,the pharmaceutical composition will be formulated with components thatwill not significantly impair the biological properties of the liveMyxoma virus, or cause degradation of or reduce the stability orefficacy of the rapamycin where included.

The pharmaceutical composition can be prepared by known methods for thepreparation of pharmaceutically acceptable compositions suitable foradministration to patients, such that an effective quantity of theactive substance or substances is combined in a mixture with apharmaceutically acceptable vehicle. Suitable vehicles are described,for example, in Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA1985). On this basis, the compositions include, albeit not exclusively,solutions of the Myxoma virus, optionally with rapamycin, in associationwith one or more pharmaceutically acceptable vehicles or diluents, andcontained in buffer solutions with a suitable pH and iso-osmotic withphysiological fluids.

The pharmaceutical composition may be administered to a patient in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. The composition of theinvention may be administered orally or parenterally. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationmay be by continuous infusion over a selected period of time.

The pharmaceutical composition may be administered orally, for example,with an inert diluent or with an assimilable carrier, or it may beenclosed in hard or soft shell gelatin capsules, or it may be compressedinto tablets. For oral therapeutic administration, the Myxoma virus maybe incorporated, optionally together with rapamycin, with an excipientand be used in the form of ingestible tablets, buccal tablets, troches,capsules, elixirs, suspensions, syrups, wafers and the like.

Solutions of Myxoma virus, optionally together with rapamycin, may beprepared in a physiologically suitable buffer. Under ordinary conditionsof storage and use, these preparations contain a preservative to preventthe growth of microorganisms, but that will not inactivate the livevirus. A person skilled in the art would know how to prepare suitableformulations. Conventional procedures and ingredients for the selectionand preparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences and in The United StatesPharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

In different embodiments, the composition is administered by injection(subcuteanously, intravenously, intramuscularly, etc.) directly at thedisease site, such as a tumour site, or by oral administration,alternatively by transdermal administration.

The forms of the pharmaceutical composition suitable for injectable useinclude sterile aqueous solutions or dispersion and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions, wherein the term sterile does not extend to the live Myxomavirus itself that is to be administered. In all cases the form must besterile and must be fluid to the extent that easy syringability exists.

The dose of the pharmaceutical composition that is to be used depends onthe particular condition being treated, the severity of the condition,the individual patient parameters including age, physical condition,size and weight, the duration of the treatment, the nature of concurrenttherapy (if any), the specific route of administration and other similarfactors that are within the knowledge and expertise of the healthpractitioner. These factors are known to those of skill in the art andcan be addressed with minimal routine experimentation.

The Myxoma virus, optionally in combination with rapamycin, orpharmaceutical compositions comprising the Myxoma virus and rapamycin,either together in the same formulation or different formulations, mayalso be packaged as a kit, containing instructions for use of Myxomavirus and rapamycin, including the use of Myxoma virus, or use of Myxomavirus in combination with rapamycin, to inhibit a cell that has adeficient innate anti-viral response, or use of Myxoma virus, or use ofMyxoma virus in combination with rapamycin, to treat a disease statecharacterized by the presence of cells that have a deficient innateanti-viral response, in a patient in need thereof. The disease state maybe cancer, or it may be a chronic viral infection.

The present invention also contemplates the use of Myxoma virus,optionally in combination with rapamycin, for inhibiting a cell that hasa deficient innate anti-viral response. In one embodiment, the cell isnon-responsive to interferon. There is further provided use of Myxomavirus, optionally in combination with rapamycin, for treating a diseasestate characterized by the presence of cells that have a deficientinnate anti-viral response, in a patient in need thereof. In oneembodiment the disease state is cancer. There is also provided use ofMyxoma virus, optionally in combination with rapamycin, in themanufacture of a medicament, for inhibiting a cell that has a deficientinnate anti-viral response, or for treating a disease statecharacterized by the presence of cells that have a deficient innateanti-viral response in a patient in need thereof.

MV can selectively infect cells in or derived from animals other thanthe natural host of MV, from a population of cells, which have adeficient innate anti-viral response. This ability of MV provides forthe use of MV in detecting cells from a population of cells, either inculture or in an animal, that have a deficient innate anti-viralresponse, including cells that are non-responsive to interferon. Suchcells may otherwise not be easily detectable, for example certain cancercells that have not yet advanced to palpable tumour, or have not yetinduced noticeable symptoms in the animal.

Thus, in one embodiment, there is provided a method for detecting cellsthat have a deficient innate anti-viral response in a patient,comprising administering to the patient Myxoma virus modified to expressa detectable marker, optionally in combination with rapamycin; allowingthe virus to infect a cell that has a deficient innate anti-viralresponse in the patient; and detecting the cell expressing thedetectable marker in the patient.

The infected cells may be detected using any conventional method forvisualizing diagnostic images. The method of detection will depend onthe particular detectable marker that is used. For example, cellsinfected with Myxoma virus genetically modified to express a fluorescentprotein may be detected using fluorescence digital imaging microscopy.Other methods include computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography. Skilled artisans will be able to determine theappropriate method for detecting a particular detectable marker.

The detectable marker includes, but is not limited to, any marker forwhich genes for its expression or synthesis can be inserted into theMyxoma genome so as to result in expression or synthesis of the markerwithin cells that are infected by the modified virus. For example, inone embodiment, the detectable marker may be a fluorescent protein. Theinfected cells may be detected at a suitable time interval afteradministration of the modified virus to the patient, so as to allow forthe virus to infect any cells that have a deficient innate anti-viralresponse, and to express the detectable marker in such cells at levelsthat would allow for detection. For example, detection may occuranywhere between 2 and 20 days following administration to the patientof the virus genetically modified to express a fluorescent protein.

The detecting method may be carried out repeatedly at intervals in apatient in order to monitor the presence of cells that have a deficientinnate anti-viral response in that patient. For example, the method fordetecting such cells using Myxoma virus may be carried out on a patientat 6 month, 1 year or 2 year intervals, as is necessary, depending onthe nature of the cells that has a deficient innate anti-viral responseand the nature of any disease state caused as a result of the presenceof such cells in a patient. Repeating the method over a time periodallows for monitoring of the progression or remission of disease state,or the spread of disease within the body of the patient.

Myxoma virus is capable of selectively infecting cells that have adeficient innate anti-viral response, and can be used as an indicator ofsuch a deficiency in cells. Thus, cells removed from a patient may beassayed for deficiency in innate anti-viral response using the methodsof the present invention. Such determination may indicate, when combinedwith other indicators, that the patient may be suffering from aparticular disease state, for example, cancer.

In one embodiment therefore, there is provided a method for detecting ina sample a cell that has a deficient innate anti-viral responsecomprising culturing the cell, exposing cultured cells to Myxoma virus,optionally in combination with rapamycin; and determining infectivity ofcells by Myxoma virus.

The cells may be removed from a subject, including a human subject,using known biopsy methods. The biopsy method will depend on thelocation and type of cell that is to be tested.

Cells are cultured according to known culturing techniques, and areexposed to MV, and optionally rapamycin, by adding live Myxoma virus,and optionally rapamycin, to the culture medium. Where Myxoma virus isadded in combination with rapamycin, the virus and rapamycin may beadded either simultaneously or sequentially. The multiplicity ofinfection (“MOI”), including in the presence of rapamycin, may be variedto determine an optimum MOI for a given cell type, density and culturetechnique, and a particular rapamycin concentration, using a positivecontrol cell culture that is known to be infected upon exposure to MV.

The amount of rapamycin, and the timing of addition of rapamycin andMyxoma virus to the cultured cells may be varied depending on cell type,method of culturing and strain of virus. Such parameters can be readilytested and adjusted with minimal testing using routine methods.

Infectivity of the cultured cells by MV, including in the presence ofrapamycin, may be determined by various methods known to a skilledperson, including the ability of the MV to cause cell death. It may alsoinvolve the addition of reagents to the cell culture to complete anenzymatic or chemical reaction with a viral expression product. Theviral expression product may be expressed from a reporter gene that hasbeen inserted into the MV genome.

In one embodiment the MV may be modified to enhance the ease ofdetection of infection state. For example, the MV may be geneticallymodified to express a marker that can be readily detected by phasecontrast microscopy, fluorescence microscopy or by radioimaging. Themarker may be an expressed fluorescent protein or an expressed enzymethat may be involved in a colorimetric or radiolabelling reaction. Inanother embodiment the marker may be a gene product that interrupts orinhibits a particular function of the cells being tested.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Infection of Mouse and Human Cell Lines with MyxomaVirus Virus Strains

Viral strains used include wildtype MV, MV modified to express eithergreen fluorescence protein (“GFP”) or β-galactosidase (“LacZ”), andkilled (“dead”) MV. Viruses were prepped and titred using standardtechniques.

Cell Strains

Mouse experiments were performed using mouse embryo fibroblasts (“MEFs”)derived from a wild-type mouse, and from the following mouse knockouts:IFNα/β receptor homozygous knockout; STAT1 homozygous knockout; PKRheterozygous; RNaseL heterozygous knockout; Mx1 heterozygous knockout;triple PKR/RNaseL/Mx1 homozygous knockout.

Human experiments were performed on BGMK control cells and human tumourcell lines HT29, HOP92, OVCAR4, OVCAR5, SK-MEL3, SK-MEL28, M14, SKOV3,PC3, DU145, CAKI-1, 786-0, T47D, MDAMB 435, SF04, U87, A172, U373, Daoyand D384 as described in Stojdl et al., Cancer Cell (2003) 4: 263-275.

Methods

Generally, assays and experiments were performed as described in Lalaniet al. Virology (1999) 256: 233-245; Johnston et al. J Virology (2003)77(13): 7682-7688; and Sypula et al. Gen Ther Mol Biol (2004) 8: 103.

For the in vivo mouse studies, nude mice were implanted withintracranial human gliomas U87. 15 days after implantation, mice wereintratumourally injected with live or dead MV GFP, at a titre of 5×10⁶,or mock-infected. 72 hours post-infection, animals were sacrificed, thebrains removed, embedded in OCT (Optimal Cutting Temperature compound),and frozen sections were cut. Myxoma-GFP was visualized in whole brainsections by fluorescence microscopy. Sections were then fixed andstained with H&E (hemotoxylin and eosin) to visualize the tumor.

For human tumour cell assays, the tumours were trypsonized and platedimmediately after surgery and infected with virus the next day at an MOIof 0.1, 1.0 or 10. Data was gathered regarding cytotoxicity and viralexpression using phase microscopy and fluorescent microscopy,respectively, at 24 and 48 hours post-infection. Assays using the yellowtetrazolium salt MTT were performed to quantify the % cell survival (asa percentage of cells surviving mock infection) at 48, 72 or 96 hourspost-infection.

Human pediatric medulloblastoma cell lines, Daoy and D384, were infectedwith 10 M.O.I. of Myxoma-GFP. 72 hours after infection, cell viabilitywas measured using MTT.

Results: Infections of Mouse Cell Lines

Previous research showed that some clones of mouse 3T3 cells transfectedwith chemokine receptors were infectable by Myxoma virus while otherclones were not. To investigate whether Myxoma virus tropism in othermouse cells was dependent on any particular receptors, we exploitedprimary mouse embryo fibroblasts (MEFs) from wild-type (WT) mice andvarious gene knock-outs.

Since IFNs play a key role in mounting anti-viral responses, wehypothesized that the restrictive phenotype was related to the“antiviral state” mediated by IFN. Disruption of the chain of events ofthe IFN system, neutralizing circulating IFN with antibodies orgenerating IFN receptor negative mice, or mice with deleted genes in theintracellular pathway of signal transmission, would severely compromisethe host's resistance to the Myxoma virus which typically does notinfect normal mouse cells.

In order to test this hypothesis we needed to demonstrate if thenon-infectivity of Myxoma virus in the nonpermissive cells was due tothe antiviral action of IFNs. Various MEF cell types having knock-outsof one or more proteins involved in intracellular IFN signaling responsewere tested for the effect of MV infection on the IFN pathway.

Experiments performed on primary MEFs demonstrated that wildtype (“WT”)MEFs are not infectable by Myxoma virus. The MEFs are fully infectableby Myxoma virus when the IFN pathway is blocked by neutralizing antibodyto IFNα/β (FIG. 2). However, MEFs exposed to neutralizing antibodies toIFNγ remained nonpermissive. This outlined the importance of IFNα/β butnot IFNγ in creating a permissive environment for Myxoma virus to infectMEFs in vitro. Different intracellular signaling pathways for IFNα/β andIFNγ have been identified in the literature. However, both IFNα/β andIFNγ likely play an important role in infected hosts, unlike culturedfibroblasts. We predict that human tumors deficient in either IFNα/βand/or IFNγ pathway will be susceptible to Myxoma virus infection invivo.

We examined the activity of STAT1 and STAT2 in nonpermissive WT MEFsthat were infected with MV. The results shown in FIG. 3 indicated thatSTAT1 and STAT2 were activated. Further study showed that STAT3, STAT4and STAT5 are not activated (FIG. 4).

In order to confirm the importance of the IFNα/β intracellular pathwayin maintaining a nonpermissive state in MEFs, genetic deletion studieswere performed to provide disruptions in the IFNα/β receptors and in theintracellular cascade. Genetic deletion of IFN receptors or JAK1 orSTAT1 was performed. MV was used to infect WT MEFs, IFNα/β R−/− MEFs andSTAT1 −/− MEFs. IFNα/β R−/− MEFs and STAT1 −/− MEFs were permissive toMV demonstrating the IFNα/β and STAT1 signalling cascades are criticalfor MV infection (FIG. 5).

Protein Kinase R (PKR) is an enzyme induced in a wide variety of cellsby IFNα/β. This kinase, in the presence of dsRNA, undergoesautophosphorylation and then phosphorylates several cellular proteinsincluding eukaryotic protein synthesis initiation factor (eIF-2α) whosephosphorylation can induces an inhibition of protein translation andapoptosis. PKR is also indicated in the activation of RNaseL. Weexamined the activation of PKR in nonpermissive MEFs following MVinfection. PKR is not phosphorylated in nonpermissive MEFs in which theantiviral state is well established (FIG. 6). Furthermore MV infectioninhibits PKR phosphorylation (FIG. 7). In addition, PERK (PKR-like, ERkinase) is not phosphorylated in the primary WT MEFs following Myxomavirus infection (FIG. 8).

MV was use to infect MEFs with single gene knockouts of PKR, RNaseL orMx1 (FIG. 9). It was discovered that PKR, RNaseL and Mx1 arenonessential for maintaining nonpermissiveness for Myxoma virusinfection. To further confirm the nonessential role of PIER, RNaseL andMx1 a Triple knockout of PKR−/−, RNase L−/− and Mx1−/− in MEFs wasperformed. A PKR−/−, RNase L−/− and Mx1−/− triple knockout does notsupport Myxoma virus infection (FIG. 10), however MEFs with a triple KOof PKR, RNaseL and Mx1 treated with a neutralizing antibody toInterferon α/β becomes permissive to Myxoma virus infection (compareFIGS. 10 and 11). These experiments demonstrate that PKR, RNaseL and Mx1are not essential in mediating the nonpermissiveness of MEFs to MV.

Further studies were performed to examine the activation of eIF-2α andPKR in nonpermissive wildtype MEFs and permissive IFNα/β R−/− MEFs andSTAT1−/− MEFs after MV infection. After MV infection, eIF-2α isphosphorylated in nonpermissive and permissive MEFs although PKR is notphosphorylated in either case (FIG. 12). This demonstrates that withoutthe involvement of PKR and PERK, the antiviral state is mediated byanother pathway that causes eIF2α phosphorylation.

STAT1 is both serine- and tyrosine-phosphorylated following Myxomainfections in nonpermissive PKR, RNaseL and Mx1 Triple KO MEFs (FIG.13). Subcellular localization of tyrosine-phosphorylated STAT1 innonpermissive PKR−/−+RNaseL−/−+Mx1 −/− MEFs following Myxoma virusinfection is also shown (FIG. 14).

In summary, these results indicate that a parallel PKR/PERK-independentantiviral pathway involving IFN/STAT1 is critical for poxvirus tropism.Furthermore, eIF2α phosphorylation is the best marker for the antiviralaction by INF.

Results: Human Tumour Studies

We studied the ability of MV to infect human tumour cells in an in vivosystem. Nude mice were injected with human glioma cells, andsubsequently developed intracranial gliomas. Live virus was able toinfect these human tumours cells but did not infect surrounding cells(FIG. 15). The localization of fluorescent signal from GFP to the tumouris depicted in FIG. 16.

Given that many human tumours are non-responsive to interferon, and thatthe tumour cells do not have normal IFN signaling cascades compared tothose found in normal human cells, studies were performed to investigatethe effect of Myxoma virus on human tumours. The results are summarizedbelow.

Initially, Myxoma virus was used to study the infectivity and cytolyticeffects on various control and human tumour cell lines: BGMK, HT29,HOP92, OVCAR4, SK-MEL3, and SK-MEL28. MV demonstrated variousinfectivity and cytolytic results: HT29 (FIG. 17) HOP92 (FIG. 18),OVCAR4 (FIG. 19) SK-MEL3 (FIG. 20), SK-MEL28 (FIG. 21) and BGMK (FIG.22).

Additional tumour cells were tested and Table 1 below classifies thevarious tumour types tested as permissive or non-permissive.

TABLE 1 Myxoma Virus Trophism for Human Tumour Cells Cell Line CellOrigin Species Permissive Non-Permissive BGMK Kidney Monkey X RK-13Kidney Rabbit X RL5 T-Lymphocyte Rabbit X HOS Osteosarcoma Human X PC3Prostate cancer Human X Caki-1 Renal cancer Human X HCT116 Colon cancerHuman X 786-0 Renal cancer Human X SK-OV-3 Ovarian cancer Human X ACHNRenal cancer Human X HOP92 Lung cancer Human X SK-MEL3 Melanoma Human XSK-MEL28 Melanoma Human X OVCAR4 Ovarian cancer Human X OVCAR5 Ovariancancer Human X DU145 Prostate cancer Human X A498 Renal cancer Human XT47D Breast cancer Human X Colo205 Colon cancer Human X HT29 Coloncancer Human X MDAMB435 Breast cancer Human X M14 Melanoma Human X MCF7Breast cancer Human X SK-MEL5 Melanoma Human X

Various human tumour lines demonstrated varying responsiveness toinfection with increasing concentrations of MV-LacZ. For example, U373cells required higher virus titres to achieve the levels of cell killingachieved with lower virus titres in U87 (FIG. 23 and FIG. 24). Myxomaefficiently infected astrocytoma cells (FIG. 25), and glioma cells (FIG.26). Myxoma was effective at 48 hours post-infection at killing humanastrocytoma and pediatric medulloblastoma cells (FIGS. 27 and 28).

Example 2 Effect of Rapamycin on the Kinetics of Myxoma VirusReplication in Restrictive Cell Lines Virus Strains

Viral strains used include wildtype MV (“vMyxLac”), and MV modified tohave the MT-5 gene knocked out (“vMyxLacT5-”). Viruses were prepped andtitred using standard techniques.

Cell Strains

Human experiments were performed on BGMK primate control cells, RK-13rabbit control cells and normal human fibroblasts A9, restrictive humantumour cell lines 786-0 (renal), ACHN (renal), HCT116 (colon), MCF-7(breast), MDA-MB-435 (breast), M14 (melanoma) and COL0205 (colon).

Methods

Generally, assays and experiments were performed as described in Lalaniet al. Virology (1999) 256: 233-245; Johnston et al. J Virology (2003)77(13): 7682-7688; and Sypula et al. Gen Ther Mol Biol (2004) 8: 103.

For viral growth curves, cells were grown in vitro in a monolayer, andpretreated with 20 nM rapamycin or a control (1:5000 dilution of DMSO)prior to infection with virus.

Samples of indicated cell lines infected with the indicated viral strainwere collected at 72 hours post infection and lysed. The virus containedwithin the cell lysates was titrated and used to infect BGMK monolayers.At 48 hours post infection, cells were fixed and stained using X-gal.

Results

Myxoma virus has been previously demonstrated by the inventors to beable to infect and replicate in many types of human tumor cells (Sypulaet al. (2004) Gene Ther. Mol. Biol. 8:103). This rabbit specific viruscan preferentially infect a majority (approximately 70%) of human cancercell lines from the NCI reference collection. In addition, the hostrange gene M-T5 was found to play a critical role during Myxoma virusinfection of many of these cell lines.

In the present investigation of potential intracellular molecules thatmay be affecting the ability of Myxoma to selectively replicate withinhuman tumour cells, the effect of rapamycin was tested.

As seen in FIG. 29, the ability of Myxoma virus to replicate and spreadfollowing a low multiplicity of infection (MOI) was performed using amultistep growth curve, using BGMK (control primate cell line); RK-13and RL5 (control rabbit cell lines); 4T1 and B16F10 (mouse cancer celllines); HOS and PC3 (permissive human cancer cell lines); 786-0, HCT116and ACHN (restrictive human cancer cell lines); MCF-7, M14 and COLO205(abortive human cancer cell lines). Both wild type vMyxLac and the M-T5knock out virus vMyxT5KO were tested to investigate the ability of bothviruses to infect and spread throughout the monolayer in the presenceand absence of pre-treatment with rapamycin. Virus titre was assessed byfoci formation on BGMK cells. Cells were pretreated with 20 nM rapamycinor appropriate vehicle control (1:5000 dilution of DMSO) for 6 hoursbefore infection.

As demonstrated, rapamycin has no effect on control BGMK cells, nor oneither of the rabbit cell lines tested, including the RL-5 cells, whichare non permissive for the MT-5 knock out virus. However, rapamycin doesenhance the replication of myxoma virus in mouse tumour cell lines, andmarginally in permissive (Type I) cell lines, such as PC-3. Rapamycinhas less of an effect on highly permissive cells such as HOS cells,likely due to the fact that such cell lines are already maximallypermissive for the Myxoma virus. The greatest effect with rapamycin wasobserved in the restrictive (Type II) cell lines (786-0, HCT116 andACHN), which are permissive for wildtype virus but non-permissive forthe vMyxT5KO strain. Some effect was seen even in abortive (Type III)cell lines MCF-7 and COLO205, although not in abortive cell line M14.

Samples of the BGMK and 786-0 infected cells were then collected andlysed, and the isolated virus was used to infect monolayers of BGMKcells (FIG. 30). Virally infected cells were visualized using X-Galstaining.

Pretreatment of tumour cells that are “restrictive” for Myxomainfection, i.e. those cells that permit the replication of the wild typeMyxoma virus but not the MT-5 knock-out virus, with rapamycin resultedin a restoration of the ability of Myxoma virus to replicate in thesecancer cell lines, which include renal, colon and ovarian cancer celllines (FIGS. 29 and 30).

In addition, the treatment with rapamycin enhanced the ability of thewild type virus to replicate in these same cells, but not control rabbitor primate cells. These results indicate that rapamycin acts to enhanceMyxoma virus infection. In addition, rapamycin appears to influence theability of cancer cells that are poorly infectable by this virus topermit virus replication.

Subsequent experiments examined the effect of rapamycin treatment onhuman tumour cells that could not support wild type Myxoma virusinfection (FIG. 31). The pretreatment had little effect on controlprimate cells or normal human fibroblasts, yet could enhance virusinfectivity in several cell lines, including the breast cancer cell lineMCF-7. As several of the human tumour cell lines remained resistant torapamycin treatment, as well as the control cell lines, it is unlikelythat rapamycin treatment could permit Myxoma virus to productivelyinfect non-transformed tissue.

Example 3 Myxoma virus M135KO Variant as an Improved Oncolytic VirusCandidate

M135R is Expressed from Myxoma Virus as an Early Gene

Myxoma virus encodes a protein (135R) identified from the sequencing ofthe MV genome (Cameron et al. Virology (1999) 264: 298-318) predicted tomimic the host IFNα/β receptor and prevent IFNα/β from triggering a hostanti-viral response (Barrett et al. Seminars in Immunology (2001)13:73-84). This prediction is based on sequence homology to the viralIFNα/β receptor homolog from vaccinia virus (B18R), which virus has beendemonstrated to employ such an immune evasion strategy (Symons et al.Cell (1995) 81:551-560). However M135R is only half the size of VV B18Rand all other IFN α/β-R homologs sequenced from poxviruses, and in allcases aligns only to the amino terminus half of poxviral IFN α/β-Rhomologs. FIG. 32 indicates the predicted structure and sequencesimilarity between M135R from MV and B18R from VV. Only the first 179amino acid residues of B18R are shown in the sequence alignment. Table 2indicates the % identity between M135R and the indicated poxviral IFNα/β-R homologs. Numbers above the diagonal represent % identity andnumbers below the diagonal represent % similarity between any twospecies. The numbers in brackets across the top represent the number ofamino acids in the putative proteins. Comparison was done between thepredicted full length copy of M135R (178 amino acids) and the first 178residues of each homolog only.

TABLE 2 Comparison of M135R to Other Poxviral Homologs % Identity MyxomaVaccinia Variola Monkeypox Cowpox Ectromelia Camelpox YLDV Swinepox LSDVspecies (178) (351) (354) (352) (351) (358) (355) (351) (344) (360)Myxoma — 24 21 24 23 21 22 20 18 17 Vaccinia 39 — 80 93 90 84 79 20 2325 Variola 36 88 — 79 87 88 79 19 24 24 Monkeypox 38 95 87 — 86 83 78 2020 26 Cowpox 38 93 93 91 — 92 88 21 23 25 Ectromelia 35 89 93 87 94 — 8717 21 25 Camelpox 34 86 94 85 92 93 — 17 23 24 YLDV 38 37 37 37 38 34 34— 23 28 Swinepox 32 39 39 36 39 35 37 38 — 25 LSDV 32 39 39 41 38 39 3643 38 —

Peptides against predicted immunogenic regions of M135R were synthesizedand used to generate polyclonal antibodies in rabbits that were used inwestern blot analysis, immuno-precipitations and immuno-fluorescence.Immunoblotting confirmed that M135R is synthesized as an early genewhose expression can be detected as early as three hours post infection(FIG. 33A; lane 1: mock infected BGMK cells; lanes 2-6: BGMK cellsinfected with vMyxLau 0, 3, 6, 18 and 36 hours post infection,respectively). Treatment of infected cells with AraC indicates thatsynthesis of M135R was not altered by inhibition of late proteinexpression and is therefore an early gene (FIG. 33B). However treatmentwith tunicamycin indicates that M135R is N-linked glycosylated, likelyat the single site predicted from the sequence (FIG. 33B). Monensintreatment suggests that there is no O-linked glycosylation. For theresults shown in FIG. 33, BGMKs were infected at an moi of 10 withMyxoma virus. Cells were treated with AraC at a concentration of 40μg/ml, tunicamycin at 1 μg/ml and monensin at 1 μg/ml, or wereuntreated, at the times indicated. M135R was detected with a peptideantibody.

M135R Encodes a Signal Sequence but is not Secreted

Sequence analysis of M135R indicates the presence of a predicted signalsequence (FIG. 32B). However there is also a predicted transmembranedomain at the carboxy terminus (FIG. 32B). Immunoblots of supernatantsfrom infected BGMK cells indicate that M135R is not secreted. However,M135R is easily detected in whole cell lysates (FIG. 33). To testwhether the signal sequence functioned to drive M135R to the cellsurface, we deleted the transmembrane domain and cloned the mutant intoa baculovirus expression system. Comparison of AcM135R and Ac135ΔTMinfected supernatants indicated that full length M135R is found in thecell lysate there is no evidence of secretion. In contrast Ac135ΔTM issecreted and confirms that the signal sequence functions to drive M135Rinto the extracellular environment (data not shown).

M135R Protein Localizes to the Surface of Infected Cells

The observation that M135R has a functional signal sequence as well as atransmembrane domain prompted us to test the localization of M135R. Twopieces of evidence indicate that M135R localizes to the cell surface.First, when BGMKs were seeded onto glass coverslips and infected withvMyxLau (moi of 10) for 24 hours then M135R was detected byimmunostaining with affinity purified anti-M135R followed byFITC-conjugated secondary antibody (FIG. 34A). M135R staining patternindicates localization to the cell surface of infected cells. vMyxLau isa true wildtype strain of Myxoma virus which has not been altered byinsertion of the β-gal or EGFP gene.

The second piece of evidence for cell surface localization M135R followsbiotinylation of cell surface proteins of GHOST cells infected witheither vMyxgfp or vMyx135KO. Twenty-four hours post infection celllysates were prepared. Streptavidin agarose beads were mixed with 500 μgof total cellular protein from cell lysates for 45 minutes. The beadswere washed and separated on a 15% PAGE-SDS gel and then probed withanti-M135R. 50 μg of total protein from the infected cell lysates wererun as controls. Immunoprecipitation of biotinylated surface proteinsindicates that m135R is at the surface of infected cells (FIG. 34B).

M135R is Non-Essential for Myxoma Virus Replication In Vitro

To test the ability of M135R to act as a virulence factor we constructeda recombinant virus in which M135R was deleted and replaced by acassette encoding EGFP and gpt under VV early/late promoters (460nucleotides, or 86% of the orf was deleted). The cloning strategy andcassette is shown in FIG. 35A. The recombinant was plaque-purified byselecting virus clones expressing EGFP. The purity of the recombinantwas confirmed by PCR (FIG. 35B; Lane 1 is the 1 Kb plus DNA ladder, Lane2 and 3 are PCR products from two purified vMyx135KO clones. The PCRproduct represents the region into which the M135R coding region hasbeen deleted and the EGFP/gpt marker has been inserted. Lane 2 is plaque1 and Lane 3 is plaque 2. Lane 4 represents the same region and coversthe native, uninterrupted M135R locus.). Immunoblotting of BGMK cellsinfected with either vMyxLau or vMyx135KO confirmed that vMyx135KO hadlost M135R expression (FIG. 35C; time course of expression of M135R:Lane 1 is uninfected BGMK cells. Lanes 2-6 represent BGMK cells infectedwith vMyxLau at times 0 (lane 2), 3 (lane 3), 6 (lane 4), 18 (lane 5),and 36 hours post infection (lane 6). Lanes 7 and 8 represent BGMK cellsinfected with vMyx135KO at 6 (lane 7) and 18 (lane 8) hours postinfection. Lane 9 is a positive control with M135R expressed in AcNPV.).

Single step growth curves were used to test the ability of vMyx135KO toreplicate in BGMK cells. BGMK cells were infected with vMyxgfp orvMyx135KO at an moi of 5 and cells were collected at the timesindicated. Virus titres were determined on BGMK cells. There was nodifference in the replication pattern between vMyxgfp and vMyx135KO(FIG. 36). These results indicate that M135R is not required forreplication in vitro.

During our studies of the ability of another gene of Myxoma to influenceMyxoma replication in rabbit primary embryo fibroblasts (REFs), we usedvMyx135KO as a knockout control and observed a curious phenomenon.Infection of the REFs with vMyxgfp resulted in a normal focus ofinfection however vMyx135KO produced a plaque-like zone of infection(FIG. 37). When we tested other cells to confirm this phenotype we wereable to replicate the plaque formation in other rabbit fibroblasts(HIG-82, FIG. 38) and human primary fibroblasts (ccd922-sk, FIG. 39).

M135R is a Critical Virulence Factor for Pathogenesis in Rabbits

We next tested the ability of vMyx135KO to produce myxomatosis in labrabbits. In contrast to the animals injected with vMyxLau or vMyxgfpwhich developed normal myxomatosis and had to be euthanized between days9 and 10 post injection, the rabbits injected with vMyx135KO recoveredcompletely (Table 3). To confirm that loss of M135R caused theattenuation of vMyx135KO we generated a revertant virus in which M135Rwas restored and we tested the ability of this revertant (vMyx135REV) torestore the ability to produce myxomatosis. All four treated groups ofrabbits responded in a similar manner for the first six days followinginjection of the respective viruses (Table 3). We observed a large, red,raised lesion at the site of injection in all treatment groups by 4 dayspost infection. However beginning at day 6 and continuing over the next3-4 days the differences between the different viruses became evident.Those animals injected with the wildtype or revertant virus had numeroussecondary lesions in the ears, eyes and nose which were not observed inthe animals injected with vMyx135KO (Table 3). We conclude that loss ofM135R drastically attenuated MV in animal models and indicates thatM135R is a critical virulence factor.

TABLE 3 Pathogenesis of vMyx135KO Compared to Wildtype ControlsObservations and Time of onset (number + days indicates first appearancein days post injection) Clinical Signs Lausanne (4 animals) vMyx135KO (6animals) vMyx135REV (3 animals) inoculation 2 days: red, visible 4 days:11-16 mm 3 days: small red lump, site slightly raised red, raised, darkslightly raised 4 days: red, dark centre centre satellites 4 days 6days: just 6 days: 5-10 visible beginning, over increasing to 30-40course of infection satellites visible by day 8 very few observedconjunctival none observed 9 days: single rabbit none observedinflammation discharge from eye anogenital 7 days: swelling 7 days:redness, edema swelling secondary 6-7 days: first around 7 days: fewsmall red 6 days: first observed as lesions eyes then ears spots not yetlesions, red areas on eyelids, ears eyes clearly lesion by day 7respiratory little or none little or none little or none difficultylesion 11 days: 25 mm, regression black, scabby satellites losing colourand becoming scabby 13 days: scab beginning to separate from healthytissue two animals all animals recovered three animals euthanized day 9euthanized day 10 two animals euthanized day 10

The temperature of rabbits was taken daily for the three days preceedingthe study. This was considered the baseline body temperatures of theanimals. We continued to take the temperatures daily of each animal forthe duration of the study. However there was no difference in bodytemperature between the treatment groups (FIG. 40). This suggests thatM135R does not play a role in the febrile response of infected animals.

M135R does not Bind or Inhibit Rabbit IFNα/β

The sequence of M135R is similar to the vaccinia B18R, an IFNα/βreceptor mimic. We tested the ability of M135R to bind rabbit type IIFN. We first iodinated rabbit IFN (5 μg, using Iodobeads) and testedthe ability of vMyx135KO infected cells to bind ¹²⁵I-rabbit IFN incomparison to cells infected with vMyxgfp (moi of 10). Cells werecollected, washed and counted in a gamma counter. Deletion of M135R didnot affect IFNα/β binding to infected cells and we did not observe anydifference in the amount of IFN bound to the cell surface of either RK13or BGMK cells (FIG. 41). As well, treatment of RK13 or BGMK cells withexogenous rabbit type1 IFN did not affect infection of cells byvMyx135KO (FIG. 42; cells were seeded in 12 well dishes and infectedwith the indicated virus at an moi of 0.01; fluorescent foci werecounted 72-96 hours post infection; 200 units of rabbit IFNα/β waseither added 24 hours prior to infection or cells were untreated). Thissame result was observed when cells were pretreated 24 h beforeinfection to induce an anti-viral state in the cell. We did not noticeany significant difference in the foci formed following infection ineither RK13 or BGMK cells (data not shown). This phenomenon was alsotrue if cells were treated with human IFNA/D (data not shown). As well,we were unable to observe any binding when Ac135ΔTM supernatants wereapplied to rabbit IFN α/β adhered to a BIAcore chip (data not shown).

Example 3 Molecular Consequences of Inhibiting mTOR in the Context ofMyxoma Virus Infection

Western Blot analysis (FIG. 43) was performed using cell lysates from786-0 cells, a Type II cancer cell line where rapamycin enhances myxomavirus infection. Lysates were collected 16 hours post infection witheither vMyxLac or vMyxT5KO at an MOI of 3, or without virus infection.Indicated lanes contain protein from cells that were pretreated with 20nM rapamycin (designated R) or appropriate vehicle control (1:5000dilution of DMSO, designated D) for 6 hours before infection. The blotswere probed using primary antibodies directed against the indicatedproteins.

As demonstrated, myxoma virus infection affects many of the signalingpathways that converge on mTOR, the physiologic target of rapamycin. Inthe context of infection with either wild type (vMyxLac) or MT-5deficient (vMyxT5KO) virus, where rapamycin has a beneficial effect onvirus replication, global effects are observed in many of thesesignaling molecules that would not be predictable based on treatmentwith rapamycin alone (see mock infected lanes). These effects include anincrease in the kinase activity of AKT-1, Raf-1, GSK-3β and mTOR itself,as well as a decrease in the kinase activity of PTEN and p70S6K. Thisdata indicate that these pathways are likely to play a role in myxomavirus permissiveness in human cancer cells lines.

As can be understood by one skilled in the art, many modifications tothe exemplary embodiments described herein are possible. The invention,rather, is intended to encompass all such modification within its scope,as defined by the claims.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. All technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art of this invention, unlessdefined otherwise.

All reference cited herein are fully incorporated by reference.

1. A method for inhibiting a cell that has a deficient innate anti-viralresponse comprising administering to the cell an effective amount of acombination of Myxoma virus and rapamycin. 2-3. (canceled)
 4. The methodof claim 1 wherein the cell is a human cancer cell.
 5. (canceled)
 6. Themethod of claim 4 wherein the Myxoma virus is genetically modified. 7.The method of claim 6 wherein the Myxoma virus is genetically modifiedto express a therapeutic gene.
 8. The method of claim 4 wherein the cellis lung cancer cell, melanoma cell, ovarian cancer cell, prostate cancercell, renal cancer cell, glioma cell or astrocytoma cell.
 9. The methodof claim 1 wherein the cell is a human cell chronically infected with avirus.
 10. A method for treating a disease state characterized by thepresence of cells that have a deficient innate anti-viral response,comprising administering to a patient in need thereof an effectiveamount of a combination of Myxoma virus and rapamycin.
 11. The method ofclaim 10 wherein the disease state is cancer.
 12. The method of claim 11wherein the cancer is a solid tumour, hematopoietic cell cancer, coloncancer, lung cancer, kidney cancer, pancreas cancer, endometrial cancer,thyroid cancer, oral cancer, ovarian cancer, laryngeal cancer,hepatocellular cancer, bile duct cancer, squamous cell carcinoma,prostate cancer, breast cancer, cervical cancer, colorectal cancer ormelanoma.
 13. The method of claim 11 wherein the cancer is lung cancer,melanoma, ovarian cancer, prostate cancer, renal cancer, glioma orastrocytoma.
 14. The method of claim 13 wherein the patient is a human.15. (canceled)
 16. The method of claim 14 wherein the Myxoma virus isgenetically modified.
 17. The method of claim 16 wherein the Myxomavirus is genetically modified to express a therapeutic gene.
 18. Themethod of claim 14 wherein the virus and the rapamycin are administeredto the site of the cancer by injection.
 19. The method of claim 14wherein the virus and the rapamycin are administered systemically. 20.The method of claim 10 wherein the disease state is a chronic viralinfection. 21-40. (canceled)
 41. A pharmaceutical composition comprisingMyxoma virus and rapamycin. 42-44. (canceled)
 45. A method for detectinga cell that has a deficient innate anti-viral response, comprisingexposing a population of cells to a combination of Myxoma virus andrapamycin; allowing the virus to infect a cell that has a deficientinnate anti-viral response; and determining the infection of any cellsof the population of cells by the Myxoma virus.
 46. The method of claim45 wherein the population of cells is in a patient; said exposingcomprises administering to the patient the combination of Myxoma virusand rapamycin, the Myxoma virus being modified to express a detectablemarker; and said determining comprises detecting a cell expressing thedetectable marker in the patient.
 47. The method of claim 45 wherein thepopulation of cells is in culture. 48-84. (canceled)
 85. A Myxoma virusthat does not express functional M135R. 86-95. (canceled)
 96. The methodof claim 1, wherein the Myxoma virus does not express functional M135R.97. The method of claim 10, wherein the Myxoma virus does not expressfunctional M135R.
 98. The pharmaceutical composition of claim 41,wherein the Myxoma virus does not express functional M135R.
 99. Themethod of claim 45, wherein the Myxoma virus does not express functionalM135R.